Novel Rat Model of Severe Cerebral Venous Sinus Thrombosis Established by Combination of Semi-Ligation, Ferric Chloride, and Thrombin

Summary

The novel rat model of severe cerebral venous sinus thrombosis (CVST), which is constructed by semi-ligation combined with ferric chloride and thrombin, can more effectively mimic the pathophysiological mechanism of severe CVST in humans.

Abstract

Cerebral venous sinus thrombosis (CVST) is a distinct type of stroke that predominantly affects young individuals, particularly pregnant women. Approximately 60% of CVST patients develop venous cerebral infarction or hemorrhage, which is defined as severe CVST. Currently, various methods are employed both domestically and internationally to induce CVST in animal models. However, these models fail to fully replicate the pathophysiological mechanisms underlying severe CVST, thereby limiting basic research on this condition. A novel rat model of severe CVST can be established through semi-ligation in combination with ferric chloride and thrombin. Semi-ligation was achieved by measuring the cerebral blood flow (CBF) in the region of interest (ROI) before and after ligation of the superior sagittal sinus (SSS) using a Perfusion Speckle Imager (PSI). Semi-ligation of the SSS induces blood stasis within the venous sinus. The topical application of ferric chloride on the surface of the SSS leads to endothelial injury, while direct injection of thrombin into the sinus creates a localized hypercoagulable state. This approach effectively mimics the three key components of thrombosis formation: stasis, endothelial damage, and hypercoagulability. The resulting model exhibits a substantial thrombotic burden involving multiple venous sinuses simultaneously. The induced thrombus remains stable for at least 1 week. The 2,3,5-triphenyltetrazolium chloride (TTC) staining demonstrates that this model can consistently produce large-area venous cerebral infarction, which persists for up to 7 days. This model can even induce epileptic seizures in rats, a capability that previous models were unable to achieve. The disruption of the blood-brain barrier was observed using Evans blue (EB) staining. Therefore, the severe CVST model established through semi-ligation combined with ferric chloride and thrombin administration more accurately replicates the pathophysiological progression of severe CVST in humans, demonstrating stability and reliability.

Introduction

Cerebral venous sinus thrombosis (CVST) is a distinct form of cerebrovascular disorder that differs from arterial stroke. It occurs more frequently in younger individuals^1,2, particularly in pregnant women³, and its incidence has been rising steadily in recent years. An epidemiological study conducted in Australia indicates that the annual incidence of CVST ranges from 13.0 to 15.7 cases per million individuals⁴. In the United States, the incidence of CVST exhibits an upward trend with age. Nevertheless, the peak age of onset for women is lower compared to that for men⁵. Between 2021 and 2023, 43 per million patients who visited emergency departments in the United States were diagnosed with CVST⁶. In clinical settings, multiple sinus thrombosis is the most commonly observed form, accounting for approximately 57.14% of all cases. This is followed by involvement of the superior sagittal sinus (SSS; 16.19%), the transverse sinus (11.43%), and the sigmoid sinus (8.57%)⁴. The main clinical manifestations include headache, which may be accompanied by epilepsy, disorders of consciousness, and focal neurological symptoms^7,8,9. However, these manifestations are not specific. Approximately 60% of patients with CVST develop cerebral venous infarction or hemorrhage, which is categorized as severe CVST^10,11. Although the treatment of CVST includes anticoagulation therapy and surgical intervention, the mortality rate of severe CVST remains as high as 34.2%⁹. This high mortality may be attributed to the fact that the underlying pathophysiological mechanisms of severe CVST are not yet fully understood. An appropriate animal model of severe CVST is therefore an essential experimental tool for investigating its pathogenesis, pathophysiological progression, and potential therapeutic strategies¹².

Currently, the methods for inducing CVST animal models can generally be categorized into the following types: permanent ligation^13,14, chemical induction^15,16,17, interventional approaches^18,19, implantation of self-made grafts^20,21,22, and bipolar electrocoagulation^23,24. The thrombus induced by previous animal models had a short duration and was unable to simultaneously induce multiple venous sinus thrombi and large-area venous cerebral infarction. Therefore, the development of a severe CVST model that involves multiple venous sinuses simultaneously has become an urgent priority. The method of semi-ligation combined with ferric chloride and thrombin can establish a novel rat model of severe CVST. Furthermore, the novel severe CVST model more accurately simulates the human pathophysiological process of severe CVST through three key aspects: venous sinus thrombus burden, venous cerebral infarction, and disruption of the blood-brain barrier. This model is applicable to research on pathophysiological mechanisms and treatment strategies for severe CVST. This novel rat model can be utilized in the future for investigating the pathophysiological mechanism of severe CVST.

Protocol

The experimental protocol was approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (AEEI-2020- 119) in Beijing, China, and was conducted in compliance with the institution's Animal Care and Use Committee regulations.

1. Animal preparation

1. Randomly assign healthy adult male Sprague-Dawley (SD) rats, with an average body weight of 300 ± 10 g, to either the experimental group or the sham operation group. All the animals were maintained on an alternating 12 h light/dark cycle with access to food and water ad libitum.

2. Application of PSI

1. Use PSI to continuously monitor alterations in cerebral blood flow (CBF) within the SSS of rats throughout the modeling process. Select a fixed circular area within the SSS as the region of interest (ROI). By monitoring the changes in CBF of the ROI during surgery, determine the completion of semi-ligation and the success of thrombus induction.

3. Establishment of severe CVST rat model

1. Anesthetize rats using 5% enflurane, with a gas mixture consisting of 70% nitrous oxide and 30% oxygen delivered via an anesthesia mask. Maintain the concentration of enflurane within the range of 1%-2% throughout the surgical procedure. Place the anesthetized rats in the prone position with their heads fixed at the midline using a stereotaxic frame. Maintain the body temperature at 37 ± 0.5 °C throughout the surgical procedure using a thermostatic heating pad.
2. Make a 1.5 cm longitudinal incision along the midline of the rat's cranial region. Dissect the subcutaneous tissue to expose the underlying skull.
3. Under microscopic visualization, make a longitudinal cranial window (10 x 4 mm) between the bregma and the lambda using a high-speed dental drill. Expose the SSS and bilateral cerebral cortex while preserving the integrity of the dura mater. During the cranial drilling process, cool the drill bit continuously with normal saline to prevent thermal injury to the dura mater and cortex.
4. Following exposure to SSS, assess the CBF of the ROI in the SSS using the PSI, and record the corresponding data. Semi-ligate the SSS rostrally and caudally using an 8-0 polyamide sutures under microscopic observation.
   NOTE: When ligating the SSS, the force should be moderate and not excessive. Otherwise, a semi-ligation effect cannot be achieved. Ligation should ideally be performed in a single continuous procedure. Repeated ligation attempts may result in perforation of the SSS, thereby compromising the success of model fabrication.
5. Utilize the PSI to assess CBF of the ROI located at the ligated segment of the SSS, confirming that the CBF had diminished to approximately 50% of that before semi-ligation. Record the results systematically.
6. Under light-protected conditions, dip a 7 mm length 3-0 silk thread in 40% ferric chloride and use it to cover the surface of the SSS ligation site for a duration of 5 min. Wash the field with normal saline after the silk thread is removed.
7. Inject 0.1 mL thrombin solution (500 U/mL) into the sinus cavity of the ligated segment using a microinjector within 1 min. Repeat the injection two additional times consecutively, for a total thrombin dose of 150 U. Thrombin reflux to the epidural space did not lead to poor thrombosis.
   1. To facilitate the injection of thrombin into the SSS, gently bend the microinjector needle prior to insertion. This adjustment allows for better control of the needle's angle during puncture and helps prevent unintended penetration through the SSS.
8. Re-evaluate the CBF of the ROI using the PSI after thrombin injection. Ensure that the results confirm a significant decrease in CBF compared to that following semi-ligation, indicating successful induction of thrombosis. Record data systematically for further analysis.
9. Seal the bone window with bone wax, followed by suturing of the incision. After the surgical procedure, administer ketoprofen (5 mg/kg) subcutaneously to each rat to alleviate postoperative pain.
10. In the sham operation group, perform the craniotomy only, without any additional surgical interventions.

4. Venous sinus thrombosis burden and cerebral infarction

1. Evaluate the burden of venous sinus thrombosis at three time points: 1 day, 2 days, and 7 days after model establishment. Sacrifice rats and perform cardiac perfusion with normal saline at different time points.
2. Conduct a craniotomy to visually assess the extent of venous sinus thrombosis involvement. Isolate the thrombus and subject to quantitative analysis. Excise the brain tissue and section it into 2 mm thick coronal slices.
3. Immerse the slices in a 2% TTC solution and incubate in a 37 °C water bath for 20 min to facilitate staining. Following this, visually assess cerebral venous infarction and quantify the infarct volume using ImageJ software for subsequent statistical analysis.
   infarct volume = area of infarct in square mm x thickness [2 mm]

5. Disruption of blood-brain barrier (BBB) permeability

1. On the second day following the surgery, randomly select rats from both the model group and the sham operation group and administer an intravenous injection of 2% Evans blue (EB) through the tail vein.
2. After 2 h of successful administration of the injection, euthanize the rats, and perfuse with phosphate-buffered saline (PBS) through the left ventricle. Harvest the brain tissues and section them into 2 mm thick slices to assess the extent of EB penetration.

6. Statistical analysis

1. Conduct statistical analysis using SPSS 17.0 software. Present measurement data as mean ± standard deviation. Use one-way analysis of variance (ANOVA) or an independent samples t-test for group comparisons.
2. For the analysis of continuous variables measured at multiple time points within the same group, use repeated measures ANOVA to assess intergroup differences across time points. A p-value of less than 0.05 was considered statistically significant.

Results

Modeling process
Figure 1A depicts the SSS of the rat, as denoted by the arrow. The rostral and caudal of the SSS were semi-ligated (Figure 1B), followed by application of ferric chloride-soaked thread onto the surface of ligated regions (Figure 1C). Thrombin was subsequently injected into the ligated segments to induce thrombosis. The area denoted by the arrow, where thrombin was injected, has turned black (

Discussion

A novel rat model of severe CVST constructed by semi-ligation combined with ferric chloride and thrombin cannot only simulate blood stasis and endothelial injury, but also the hypercoagulable state of blood, that is, it simulates the three elements of thrombosis formation²⁵. What is even more exciting is that the rat model developed using this method is capable of exhibiting epileptic seizures. Epileptic seizures represent a notable clinical manifestation in patients with CVST, occurring in approx...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,3,5-triphenyltetrazolium chloride	Sigma-Aldrich
40% ferric chloride	Tianjin Zhiyuan Chemical Reagent Co., Ltd., China
Evans blue	Sigma-Aldrich
Heating pad	Harvard Apparatus Holliston, MA, USA	50-7061-f,
High-speed dental drill	Saeshin, Busan, South Korea	Strong-207B
Laser speckle meter	PeriCam PSI System, Sweden
Microscope	Carl Zeiss, Inc., Berlin, Germany
Polyamide suture	Ningbo Chenghe Micro Apparatus Factory, China
Stereotaxic frame	David Kopf Instruments, Tujunga, California, USA
Thrombin	Chang Chun Lei Yunshang Pharmaceutical Co., Ltd., China